Neuroanatomical studies reveal that within the mass of dendrites, axons and their branches that constitute a neuropil resides a logically ordered and largely invariant set of cellular connections that interpret environmental and physiological cues and in response execute specific behaviors. In flies and vertebrates the stereotyped expression patterns of different combinations of transcription factors in partially overlapping sets of neurons ensures that neurons born at the same time and place differentiate in the same way. Thus, the precise control of transcription factor expression profiles underlies the reproducible organization of neuronal connections. However, the regulatory mechanisms that establish these patterns of transcription factor expression in neurons and the identity of the downstream target genes through which these transcription factors guide neuronal differentiation remain elusive. Our research seeks to clarify the genetic and molecular basis through which combinations of transcription factors control neuronal differentiation. Specifically, in Aim I we combine lineage and expression analyses to define the lineage and axonal projections of neurons that express hb9, Iim3 or islet, three conserved transcription factors expressed in roughly 50 neurons. In Aim II, we combine molecular and computational approaches to delimit the regulatory regions of hb9, Iim3 and islet and identify their trans-acting upstream regulators. Finally, in Aim III, we search for downstream targets of Hb9, Lim3 and Islet using genomic and classical genetic approaches. hb9, Iim3 and islet and their functions are conserved between flies and vertebrates. Thus, we expect our focused and systematic approach to reveal basic insight into the molecules and mechanisms that establish reproducible profiles of transcription factor expression in neurons as well as those through which these factors instruct neuronal differentiation.